Behavioral and neuroanatomical outcomes following altered serotonin expression in a hypoxic-ischemic injury neonate rodent model.

BACKGROUND: Children born prematurely (<37 gestational weeks) are at risk for a variety of adverse medical events. They may experience ischemic and/or hemorrhagic events leading to negative neural sequelae. They are also exposed to repeated stressful experiences as part of life-saving care within the neonatal intensive care unit (NICU). These experiences have been associated with methylation of SLC6A4, a gene which codes for serotonin transport proteins, and is associated with anxiety, depression, and increased incidence of autism spectrum disorders.The purpose of this study was to examine the effects of altered serotonin levels on behavioral and neuroanatomical outcomes in a neonatal rodent model with or without exposure to hypoxic-ischemic (HI) injury. METHODS: Wistar rat pups were randomly assigned to either HI injury or sham groups. Pups within each group were treated with a chronic SSRI (Citalopram HBr) to simulate the effects of SLC6A4 methylation, or saline (NS). Subjects were assessed on behavioral tasks and neuropathologic indices. RESULTS: HI injured subjects performed poorly on behavioral tasks. SSRI subjects did not display significantly greater anxiety. HI + SSRI subjects learned faster than HI+NS. Histologically, SSRI subjects had predominantly larger brain volumes than NS. CONCLUSION: SSRI treated subjects without injury showed patterns of increased anxiety, consistent with theories of SLC6A4 methylation. The paradoxical trend to improved cognition in HI+SSRI subjects relative to HI alone, may reflect an unexpected SSRI neuroprotective effect in the presence of injury, and may be related to serotonin-induced neurogenesis.

Evaluation of visual motion perception ability in mice with knockout of the dyslexia candidate susceptibility gene Dcdc2.

Developmental dyslexia is a heritable disability characterized by difficulties in learning to read and write. The neurobiological and genetic mechanisms underlying dyslexia remain poorly understood; however, several dyslexia candidate risk genes have been identified. One of these candidate risk genes-doublecortin domain containing 2 (DCDC2)-has been shown to play a role in neuronal migration and cilia function. At a behavioral level, variants of DCDC2 have been associated with impairments in phonological processing, working memory and reading speed. Additionally, a specific mutation in DCDC2 has been strongly linked to deficits in motion perception-a skill subserving reading abilities. To further explore the relationship between DCDC2 and dyslexia, a genetic knockout (KO) of the rodent homolog of DCDC2 (Dcdc2) was created. Initial studies showed that Dcdc2 KOs display deficits in auditory processing and working memory. The current study was designed to evaluate the association between DCDC2 and motion perception, as these skills have not yet been assessed in the Dcdc2 KO mouse model. We developed a novel motion perception task, utilizing touchscreen technology and operant conditioning. Dcdc2 KOs displayed deficits on the Pairwise Discrimination task specifically as motion was added to visual stimuli. Following behavioral assessment, brains were histologically prepared for neuroanatomical analysis of the lateral geniculate nucleus (LGN). The cumulative distribution showed that Dcdc2 KOs exhibited more small neurons and fewer larger neurons in the LGN. Results compliment findings that DCDC2 genetic alteration results in anomalies in visual motion pathways in a subpopulation of dyslexic patients.

Behavioral and histological outcomes following neonatal HI injury in a preterm (P3) and term (P7) rodent model.

Hypoxia-ischemia (HI) occurs when blood and/or oxygen delivery to the brain is compromised. HI injuries can occur in infants born prematurely (<37 weeks gestational age) or at very low birth weight (<1500 g), as well as in term infants with birth complications. In both preterm and term HI populations, brain injury is associated with subsequent behavioral deficits. Neonatal HI injury can be modeled in rodents (e.g., the Rice-Vannucci method, via cautery of right carotid followed by hypoxia). When this injury is induced early in life (between postnatal day (P)1-5), neuropathologies typical of human preterm HI are modeled. When injury is induced later (P7-12), neuropathologies typical of those seen in HI term infants are modeled. The current study sought to characterize the similarities/differences between outcomes following early (P3) and late (P7) HI injury in rats. Male rats with HI injury on P3 or P7, as well as sham controls, were tested on a variety of behavioral tasks in both juvenile and adult periods. Results showed that P7 HI rats displayed deficits on motor learning, rapid auditory processing (RAP), and other learning/memory tasks, as well as a reduction in volume in various neuroanatomical structures. P3 HI animals showed only transient deficits on RAP tasks in the juvenile period (but not in adulthood), yet robust deficits on a visual attention task in adulthood. P3 HI animals did not show any significant reductions in brain volume that we could detect. These data suggest that: (1) behavioral deficits following neonatal HI are task-specific depending on timing of injury; (2) P3 HI rats showed transient deficits on RAP tasks; (3) the more pervasive behavioral deficits seen following P7 HI injury were associated with substantial global tissue loss; and (4) persistent deficits in attention in P3 HI subjects might be linked to neural connectivity disturbances rather than a global loss of brain volume, given that no such pathology was found. These combined findings can be applied to our understanding of differing long-term outcomes following neonatal HI injury in premature versus term infants.

Bilateral subcortical heterotopia with partial callosal agenesis in a mouse mutant.

Cognition and behavior depend on the precise placement and interconnection of complex ensembles of neurons in cerebral cortex. Mutations that disrupt migration of immature neurons from the ventricular zone to the cortical plate have provided major insight into mechanisms of brain development and disease. We have discovered a new and highly penetrant spontaneous mutation that leads to large nodular bilateral subcortical heterotopias with partial callosal agenesis. The mutant phenotype was first detected in a colony of fully inbred BXD29 mice already known to harbor a mutation in Tlr4. Neurons confined to the heterotopias are mainly born in midgestation to late gestation and would normally have migrated into layers 2-4 of overlying neocortex. Callosal cross-sectional area and fiber number are reduced up to 50% compared with coisogenic wildtype BXD29 substrain controls. Mutants have a pronounced and highly selective defect in rapid auditory processing. The segregation pattern of the mutant phenotype is most consistent with a two-locus autosomal recessive model, and selective genotyping definitively rules out the Tlr4 mutation as a cause. The discovery of a novel mutation with strong pleiotropic anatomical and behavioral effects provides an important new resource for dissecting molecular mechanisms and functional consequences of errors of neuronal migration.

Evaluation of the therapeutic benefit of delayed administration of erythropoietin following early hypoxic-ischemic injury in rodents.

Hypoxia-ischemia (HI) and associated brain injuries are seen in premature as well as term infants with birth complications. The resulting impairments involve deficits in many cognitive domains, including language development. Poor rapid auditory processing is hypothesized to be one possible underlying factor leading to subsequent language delays. Mild hypothermia treatment for HI injuries in term infants is widely used as an intervention but can be costly and time consuming. Data suggest that the effectiveness of hypothermia treatment following HI injury declines beyond 6 h following injury. Consequently, the availability of a therapeutic alternative without these limitations could allow doctors to treat HI-injured infants more effectively and thus reduce deleterious cognitive and language outcomes. Evidence from both human studies and animal models of neonatal HI suggests that erythropoietin (Epo), an endogenous cytokine hormone, may be a therapeutic agent that can ameliorate HI brain injury and preserve subsequent cognitive development and function. The current study sought to investigate the therapeutic effectiveness of Epo when administered immediately after HI injury, or delayed at intervals following the injury, in neonatal rodents. Rat pups received an induced HI injury on postnatal day 7, followed by an intraperitoneal injection of Epo (1,000 U/kg) immediately, 60 min, or 180 min following induction of injury. Subjects were tested on rapid auditory processing tasks in juvenile (P38-42) and adult periods (P80-85). Ventricular and cortical size was also measured from post mortem tissue. Results from the current study show a therapeutic benefit of Epo when given immediately following induction of HI injury, with diminished benefit from a 60-min-delayed injection of Epo and no protection following a 180-min-delayed injection. The current data thus show that the effectiveness of a single dose of Epo in ameliorating auditory processing deficits following HI injury decreases precipitously as treatment is delayed following injury. These data may have important implications for experimental human neonatal intervention with Epo.

Inhibition of X-linked inhibitor of apoptosis with embelin differentially affects male versus female behavioral outcome following neonatal hypoxia-ischemia in rats.

Hypoxia-ischemia (HI; concurrent oxygen/blood deficiency) and associated encephalopathy represent a common cause of neurological injury in premature/low-birth-weight infants and term infants with birth complications. Resulting behavioral impairments include cognitive and/or sensory processing deficits, as well as language disabilities, and clinical evidence shows that male infants with HI exhibit more severe cognitive deficits compared to females with equivalent injury. Evidence also demonstrates activation of sex-dependent apoptotic pathways following HI events, with males preferentially activating a caspase-independent cascade of cell death and females preferentially activating a caspase-dependent cascade following neonatal hypoxic and/or ischemic insults. Based on these combined data, the 'female protection' following HI injury may reflect the endogenous X-linked inhibitor of apoptosis (XIAP), which effectively binds effector caspases and halts downstream cleavage of effector caspases (thus reducing cell death). To test this theory, the current study utilized neonatal injections of vehicle or embelin (a small molecule inhibitor of XIAP) in male and female rats with or without induced HI injury on postnatal day 7 (P7). Subsequent behavioral testing using a clinically relevant task revealed that the inhibition of XIAP exacerbated HI-induced persistent behavioral deficits in females, with no effect on HI males. These results support sex differences in mechanisms of cell death following early HI injuries, and suggest a potential clinical benefit from the development of sex-specific neuroprotectants for the treatment of HI.

Reprint of "Early testosterone modulated sex differences in behavioral outcome following neonatal hypoxia ischemia in rats".

Hypoxia ischemia (HI; reduced blood oxygenation and/or flow to the brain) represents one of the most common injuries for both term and preterm/very low birth weight (VLBW) infants. These children experience elevated incidence of cognitive and/or sensory processing disabilities, including language based learning disabilities. Clinical data also indicate more substantial long-term deficits for HI injured male babies as compared to HI injured females. Previously, we reported significant deficits in rapid auditory processing and spatial learning in male rats with postnatal day 1 (P1), P7, or P10 HI injury. We also showed sex differences in HI injured animals, with more severe deficits in males as compared to females. Given these findings, combined with extant clinical data, the current study sought to assess a putative role for perinatal testosterone in modulating behavioral outcome following early hypoxic-ischemic injury in rats. Male, female, and testosterone-propionate (TP) treated females were subjected to P7 HI or sham surgery, and subsequently (P30+) underwent a battery of auditory testing and water maze assessment. Results confirm previous reports of sex differences following HI, and add new findings of significantly worse performance in TP-treated HI females compared to vehicle treated HI females. Post mortem anatomic analyses showed consistent effects, with significant brain weight decreases seen in HI male and TP-treated HI females but not female HI or sham groups. Further neuromorphometric analysis of brain structures showed that HI male animals exhibited increased pathology relative to HI females as reflected in ventricular enlargement. Findings suggest that neonatal testosterone may act to enhance the deleterious consequences of early HI brain injury, as measured by both neuropathology and behavior.

Early testosterone modulated sex differences in behavioral outcome following neonatal hypoxia ischemia in rats.

Hypoxia ischemia (HI; reduced blood oxygenation and/or flow to the brain) represents one of the most common injuries for both term and preterm/very low birth weight (VLBW) infants. These children experience elevated incidence of cognitive and/or sensory processing disabilities, including language based learning disabilities. Clinical data also indicate more substantial long-term deficits for HI injured male babies as compared to HI injured females. Previously, we reported significant deficits in rapid auditory processing and spatial learning in male rats with postnatal day 1 (P1), P7, or P10 HI injury. We also showed sex differences in HI injured animals, with more severe deficits in males as compared to females. Given these findings, combined with extant clinical data, the current study sought to assess a putative role for perinatal testosterone in modulating behavioral outcome following early hypoxic-ischemic injury in rats. Male, female, and testosterone-propionate (TP) treated females were subjected to P7 HI or sham surgery, and subsequently (P30+) underwent a battery of auditory testing and water maze assessment. Results confirm previous reports of sex differences following HI, and add new findings of significantly worse performance in TP-treated HI females compared to vehicle treated HI females. Post mortem anatomic analyses showed consistent effects, with significant brain weight decreases seen in HI male and TP-treated HI females but not female HI or sham groups. Further neuromorphometric analysis of brain structures showed that HI male animals exhibited increased pathology relative to HI females as reflected in ventricular enlargement. Findings suggest that neonatal testosterone may act to enhance the deleterious consequences of early HI brain injury, as measured by both neuropathology and behavior.

Persistent spatial working memory deficits in rats following in utero RNAi of Dyx1c1.

Disruptions in the development of the neocortex are associated with cognitive deficits in humans and other mammals. Several genes contribute to neocortical development, and research into the behavioral phenotype associated with specific gene manipulations is advancing rapidly. Findings include evidence that variants in the human gene DYX1C1 may be associated with an increased risk of developmental dyslexia. Concurrent research has shown that the rat homolog for this gene modulates critical parameters of early cortical development, including neuronal migration. Moreover, recent studies have shown auditory processing and spatial learning deficits in rats following in utero transfection of an RNA interference (RNAi) vector of the rat homolog Dyx1c1 gene. The current study examined the effects of in utero RNAi of Dyx1c1 on working memory performance in Sprague-Dawley rats. This task was chosen based on the evidence of short-term memory deficits in dyslexic populations, as well as more recent evidence of an association between memory deficits and DYX1C1 anomalies in humans. Working memory performance was assessed using a novel match-to-place radial water maze task that allows the evaluation of memory for a single brief (∼4-10 seconds) swim to a new goal location each day. A 10-min retention interval was used, followed by a test trial. Histology revealed migrational abnormalities and laminar disruption in Dyx1c1 RNAi-treated rats. Dyx1c1 RNAi-treated rats exhibited a subtle, but significant and persistent impairment in working memory as compared to Shams. These results provide further support for the role of Dyx1c1 in neuronal migration and working memory.

Impaired detection of variable duration embedded tones in ectopic NZB/BINJ mice.

Utilizing rodent models, prior research has demonstrated a significant association between focal neocortical malformations (i.e. induced microgyria, molecular layer ectopias), which are histologically similar to those observed in human dyslexic brains, and rate-specific auditory processing deficits as seen in language impaired populations. In the current study, we found that ectopic NZB/BINJ mice exhibit significant impairments in detecting a variable duration 5.6 kHz tone embedded in a 10.5 kHz continuous background, using both acoustic reflex modification and auditory event-related potentials (AERP). The current results add further support to the association between focal cortical malformations and impaired auditory processing, and the notion that these auditory effects may occur regardless of the cortical location of the anomaly.

Impaired processing of complex auditory stimuli in rats with induced cerebrocortical microgyria: An animal model of developmental language disabilities.

Individuals with developmental language disabilities, including developmental dyslexia and specific language impairment (SLI), exhibit impairments in processing rapidly presented auditory stimuli. It has been hypothesized that these deficits are associated with concurrent deficits in speech perception and, in turn, impaired language development. Additionally, postmortem analyses of human dyslexic brains have revealed the presence of focal neocortical malformations such as cerebrocortical microgyria. In an initial study bridging these research domains, we found that male rats with induced microgyria were impaired in discriminating rapidly presented auditory stimuli. In order to further assess this anatomical- behavioral association, we designed two experiments using auditory-reflex modification. These studies were intended to assess whether auditory processing deficits in microgyric male rats would be seen in threshold detection of a silent gap in white noise, and in oddball detection of a two-tone stimulus of variable duration. Results showed no differences between sham and microgyric subjects on gap detection, but did show that microgyric subjects were impaired in the discrimination of two-tone stimuli presented in an oddball paradigm. This impairment was evident for stimuli with total duration of 64 msec or less, while both groups were able to discriminate stimuli with duration of 89 msec or greater. The current results further support the relationship between malformations of the cerebral cortex and deficits in rapid auditory processing. They also suggest that the parameters characterizing rapid auditory processing deficits for a specific task may be influenced by stimulus features and/or cognitive demand of that particular task.

Neonatal estrogen blockade prevents normal callosal responsiveness to estradiol in adulthood.

The rat corpus callosum (CC) is larger in males than females, and is responsive to hormone manipulations during development. We previously demonstrated that P25 ovariectomy (Ovx) enlarged (defeminized) adult CC, while P70 ovary transfer (OvT) counteracted this enlarging effect, resulting in smaller (feminized) CC. Since OvT females were not Ovx'd until P25, they received some neonatal estrogen (E) exposure. Behavioral data suggest that adult responsiveness to ovarian hormones depends upon prior organization by neonatal E. It has not been determined whether a similar phenomenon occurs for the feminization of brain morphology. The current experiment examined whether our previous finding of adult CC responsiveness to ovarian hormones depended upon neonatal E exposure. We investigated this by assessing the effects of P70 ovarian hormone replacement (via ovary transfer or E pellet) in females that received either (1) normal ovarian hormone exposure until P25 Ovx, or (2) the E receptor blocker tamoxifen from birth to P25 Ovx. Females receiving normal neonatal hormone exposure responded to P70 E in the female-typical manner: E reduced CC size. In contrast, females receiving neonatal E blockade responded to adult E in the opposite manner: E increased CC size. As far as we are aware, this is the first report suggesting that neonatal E exposure organizes the female brain so that it responds normally to the organizing actions of E when later exposure occurs. These findings further challenge the traditional model of female brain development, which asserts that normal female brain organization occurs by default, in the absence of gonadal hormone exposure.

Impaired two-tone processing at rapid rates in male rats with induced microgyria.

We previously reported that adult male rats with bilateral induced microgyria exhibit deficits in rapid auditory processing, which appear similar to auditory processing deficits seen in individuals with developmental language disabilities. The current study was designed to further elaborate that finding using an improved paradigm in which stimulus duration was uncoupled from testing experience and learning effects. Specifically, two-tone stimuli with durations of 540, 390, 332 and 249 ms were all presented within a single test session in a modified operant conditioning paradigm. Subjects were tested over a period of 12 days using this variable-stimulus format. Results confirmed microgyric male rats were impaired only in processing two-tone stimuli presented at rapid rates (i.e., 249 ms duration). Thus the current results support the previously observed link between focal malformations and deficits in rapid auditory processing.

Perceptual auditory gap detection deficits in male BXSB mice with cerebrocortical ectopias.

Underlying impairments in rapid auditory processing may contribute to disrupted phonological processing, which in turn characterizes developmental language impairment (LI). Identification of a neurobiological feature of LI that is associated with auditory deficits would further support this model. Accordingly, we found that adult male rats with induced cortical malformations were impaired in rapid auditory processing. Since 40-60% of BXSB mice exhibit spontaneous focal cerebrocortical ectopias (as seen in dyslexics brains), we assessed auditory gap detection in adult male BXSB mice. Ectopic mice were significantly worse than non-ectopics in detecting a 5 ms silent gap, but were not significantly impaired at longer gap durations (10-100 ms). Our results confirm that focal cortical malformations are associated with impairments in rapid auditory processing.

Laterality in animals: relevance to schizophrenia.

Anomalies in the laterality of numerous neurocognitive dimensions associated with schizophrenia have been documented, but their role in the etiology and early development of the disorder remain unclear. In the study of normative neurobehavioral organization, animal models have shed much light on the mechanisms underlying and the factors affecting adult patterns of both functional and structural asymmetry. Nonhuman species have more recently been used to investigate the environmental, genetic, and neuroendocrine factors associated with developmental language disorders in humans. We propose that the animal models used to study the basis of lateralization in normative development and language disorders such as dyslexia could be modified to investigate lateralized phenomena in schizophrenia.

Sex and hemispheric differences for rapid auditory processing in normal adults.

Previous research suggests that left hemisphere specialisation for processing speech may specifically depend on rate-specific parameters, with rapidly successive or faster changing acoustic stimuli (e.g. stop consonant-vowel syllables) processed preferentially by the left hemisphere. The current study further investigates the involvement of the left hemisphere in processing rapidly changing auditory information, and examines the effects of sex on the organisation of this function. Twenty subjects participated in an auditory discrimination task involving the target identification of a two-tone sequence presented to one ear, paired with white noise to the contralateral ear. Analyses demonstrated a right ear advantage for males only at the shorter interstimulus interval durations (mean = 20 msec) whereas no ear advantage was observed for women. These results suggest that the male brain is more lateralised for the processing of rapidly presented auditory tones, specifically at shorter stimulus durations.

A role for ovarian hormones in sexual differentiation of the brain.

Historically, studies of the role of endogenous hormones in developmental differentiation of the sexes have suggested that mammalian sexual differentiation is mediated primarily by testicular androgens, and that exposure to androgens in early life leads to a male brain as defined by neuroanatomy and behavior. The female brain has been assumed to develop via a hormonal default mechanism, in the absence of androgen or other hormones. Ovarian hormones have significant effects on the development of a sexually dimorphic cortical structure, the corpus callosum, which is larger in male than in female rats. In the females, removal of the ovaries as late as Day 16 increases the cross-sectional area of the adult corpus callosum. Treatment with low-dose estradiol starting on Day 25 inhibits this effect. Female callosa are also enlarged by a combination of daily postnatal handling and exogenous testosterone administered prior to Day 8. The effects of androgen treatment are expressed early in development, with males and testosterone-treated females having larger callosa than control females as early as Day 30. The effects of ovariectomy do not appear until after Day 55. These findings are more consistent with other evidence of a later sensitive period for ovarian feminization as compared to androgenic masculinization.

Cerebral microgyria, thalamic cell size and auditory temporal processing in male and female rats.

Induction of microgyria by freezing injury to the developing somatosensory cortex of neonatal rats causes a defect in fast auditory processing in males, but not in females. It was speculated that early damage to the cortex has sexually dimorphic cascading effects on other brain regions mediating auditory processing, which can lead to the observed behavioral deficits. In the current series of experiments, bilateral microgyri were induced by placement of a freezing probe on the skulls of newborn male and female rats, and these animals were tested in adulthood for auditory temporal processing. Control animals received sham surgery. The brains from these animals were embedded in celloidin, cut in the coronal plane and the following morphometric measures assessed: microgyric volume, medial geniculate nucleus (MGN) volume, cell number, and cell size, and, as a control, dorsal lateral geniculate nucleus (dLGN) volume, cell number and cell size. There were no sex differences in the cortical pathology of lesioned animals. However, microgyric males had more small and fewer large neurons in the MGN than their sham-operated counterparts, whereas there was no difference between lesioned and sham-operated females. There was no effect on dLGN cell size distribution in either sex. Microgyric males were significantly impaired in fast auditory temporal processing when compared to control males, whereas lesioned females exhibited no behavioral deficits. These results suggest that early injury to the cerebral cortex may have different effects on specific thalamic nuclei in males and females, with corresponding differences in behavioral effects.

Effects of sex and MK-801 on auditory-processing deficits associated with developmental microgyric lesions in rats.

Neonatally induced microgyric lesions produce defects in rapid auditory processing in adult male rats. Given that females across species are less susceptible to the deleterious effects of neural injury and that treatment with neuroprotective agents at the time of injury can reduce neural damage, the authors tested the effects of sex and neuroprotectant exposure on the behavioral consequences of microgyric lesions in rats. Results showed that sham but not microgyric males were able to perform the task at the fastest rate of stimulus presentation. Microgyric females, in contrast, discriminated at all stimulus conditions and did not differ from female shams. Microgyric males treated with MK-801 had reduced cortical damage and performed the discrimination at the fastest condition. Results suggest that females are less susceptible to the behavioral effects of neocortical microgyria and that MK-801 may ameliorate the behavioral consequences of these lesions in male rats.

Neurobiology of speech perception.

The mechanisms by which human speech is processed in the brain are reviewed from both behavioral and neurobiological perspectives. Special consideration is given to the separation of speech processing as a complex acoustic-processing task versus a linguistic task. Relevant animal research is reviewed, insofar as these data provide insight into the neurobiological basis of complex acoustic processing in the brain.